Protein ubiquitination is a post-translational modification used in many intracellular signaling processes. The U-box protein CHIP functions as an E3 ubiquitin ligase that catalyzes poly-ubiquitination of a wide range of misfolded proteins, which targets them for degradation in the proteasome. The ability to ubiquitinate such a large number of proteins is possible because CHIP uses heat shock proteins such as Hsc70 and Hsp70 as adaptors to recruit the misfolded substrates. The interaction with heat shock proteins makes CHIP an integral part of the protein triage system; CHIP is not only able to target a misfolded substrate for degradation via poly-ubiquitination, it can also facilitate protein folding/refolding through stabilization of heat shock protein chaperone complexes. There is a great need to understand the molecular details of how CHIP functions under cell stress and non-stress conditions given the importance of protein triage for maintaining the integrity of the proteome. The physiological importance of CHIP is further underscored by its association with neurodegenerative diseases and certain cancers through defects in the action of CHIP on specific substrates. While poly-ubiquitination by CHIP leading to targeting of substrates to the proteasome has been extensively characterized, there is a complex network of CHIP activities outside of this paradigm. Recent studies have shown that in the absence of substrates, CHIP ubiquitinates Hsc70 and Hsp70, but remarkably the downstream effect on them is different: the majority of Hsp70 is degraded in the proteasome, whereas levels of Hsc70 are only slightly lowered. We hypothesize a correlation exists between this differential regulation of the constitutive heat shock protein Hsc70 versus the stress-induced Hsp70 and differences in the ubiquitination sites and chain linkages formed. Despite the availability of structures of CHIP, E2 ubiquitin conjugating enzymes and heat shock proteins, the factors controlling the selection of ubiquitination sites on the substrate and the nature of the chain linkage formed are not known. In this proposal, we will pursue coupled biochemical and structural approaches to determine how CHIP differentially regulates Hsc70 and Hsp70. To achieve this goal, we will: (i) determine which E2 ligases support CHIP ubiquitination of Hsc70 and Hsp70; (ii) identify the corresponding sites of attachment and chain linkages formed; (iii) structurally characterize the CHIP ubiquitination machinery. These results will provide a basis for understanding the regulation of CHIP heat shock proteins, and valuable information about the mechanism for how chaperone activities are shifted in normal physiology and disease pathology.